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Haliotis Asinina) in Coastal Waters of Thailand Determined Using Microsatellite Markers

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Haliotis Asinina) in Coastal Waters of Thailand Determined Using Microsatellite Markers Mar. Biotechnol. 6, 604–611, 2004 DOI: 10.1007/s10126-004-2300-5 Ó 2005 Springer Science+Business Media, Inc. Population Structure of Tropical Abalone (Haliotis asinina) in Coastal Waters of Thailand Determined Using Microsatellite Markers S. Tang,1 A. Tassanakajon,1 S. Klinbunga,2 P. Jarayabhand,3,4 and P. Menasveta2,4 1Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand 2Marine Biotechnology Research Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathumthani 12120, Thailand 3Aquatic Resources Research Institute, Chulalongkorn University, Bangkok 10330, Thailand 4Department of Marine Science, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand Abstract: Three partial genomic libraries were constructed from genomic DNA of the tropical abalone (Haliotis asinina) that was digested with AluI, vortexed/sonicated, and digested with mixed enzyme (AluI, HincII, and RsaI). The libraries yielded 0.02%, 0.42%, and 1.46% positive microsatellite-containing clones, respectively. Eleven clones each of perfect, imperfect, and compound microsatellites were isolated. Ten primer pairs (CU- Has1–CUHas10) were analyzed to evaluate their polymorphic level. The numbers of alleles per locus, observed heterozygosity (H0), and expected heterozygosity (He) ranged from 3 to 26 alleles, and varied between 0.27 and 0.85 and between 0.24 and 0.93, respectively. Three microsatellite loci (CUHas2, CUHas3, and CUHas8) were further used for examination of genetic diversity and differentiation of natural H. asinina in coastal waters of Thailand. Genetic variabilities in terms of the effective number of alleles (ne), H0, and He were higher in 2 samples from the Gulf of Thailand (ne = 9.37, 7.66; H0 = 0.62, 0.78; and He = 0.87, 0.86) than those of one sample (ne = 6.04; H0 = 0.58; and He = 0.62) derived from the Andaman Sea. Assessment of genetic hetero- geneity, including allele frequency comparison and pairwise FST analysis, indicated interpopulational differ- entiation, between natural H. asinina from the Gulf of Thailand and that from the Andaman Sea (P < 0.0001). Key words: abalone, Haliotis asinina, microsatellites, genetic diversity, population differentiation. INTRODUCTION 1996). Abalone aquaculture has been established in several countries, but approximately 75% of the world production Abalones are marine gastropods distributed worldwide annually is in China (mainly Haliotis discus hannai) and along the coastal waters of tropical and temperate areas Taiwan (mainly H. diversicolor supertexta) (Gordon, 2000). (Geiger, 2000). Approximately 20 species of abalone are Three species of tropical abalone, H. asinina, H. ovina, commercially important (Jarayabhand and Paphavasit, and H. varia, are locally found in Thai waters (Jarayabhand and Paphavasit, 1996). Among these species H. asinina is Received June 5, 2003; accepted February 18, 2004; online publication March 9, 2005. the most promising for aquaculture. Artificially propagated Corresponding author: A. Tassanakajon; e-mail: [email protected] breeding programs and culture techniques for H. asinina Microsatellite polymorphism in H. asinina 605 are well developed; however, basic information on genetic population differentiation and levels of genetic diversity of H. asinina is necessary to improve the stock selection program and conserve the existing natural gene pool (Klinbunga et al., 2003). An initial step toward natural stock management of H. asinina is to develop molecular genetic markers that can be applied to its genetic management, including determi- nation of stock structure, evaluation of the levels of gene flow, and reconstruction of intraspecific phylogeny. In addition, the ability to determine individuality and par- entage of H. asinina will provide the means to establish efficient selective breeding programs and to construct ge- netic linkage maps of H. asinina more effectively. Microsatellites are short, tandemly repeated DNA loci (1–6 nucleotides) arrayed for approximately 10 to 50 copies and abundantly dispersed in eukaryotic genomes. Micro- satellite loci exhibiting large numbers of alleles are ideally suited for gene mapping and pedigree analysis (Pepin et al., 1995), whereas loci with lower polymorphic levels can be used for population genetic studies (Wright and Bentzen, 1994). Microsatellite markers have been successfully devel- oped in several abalone species including H. rufescens (Kirby and Powers, 1998), H. rubra (Huang and Hana, 1998; Evans et al., 2001), H. asinina (Selvamani et al., 2000), H. discus discus (Sekino and Hara, 2001), and H. discus hannai and H. kamtschatkara (Miller et al., 2001). Genetic diversity and population differentiation of abalone in Thai waters based on microsatellite polymor- Figure 1. Map of Thailand indicating sampling sites of H. asinina phism have not been reported. The objective of this study used in this study. Dots represent sample locations (except the was to develop informative microsatellites in H. asinina and Philippines sample) from which H. asinina was collected. CAM to assess the genetic structure of this species in coastal indicates Cambodia; SAM, Samet Island; TRA, Trang. waters of Thailand. We isolated H. asinina microsatellites from 3 partial genomic libraries that were constructed bodia (HACAME, N = 23) located in the Gulf of Thailand. using different approaches. Genetic heterogeneity among Samples of hatcheries were offspring of wild broodstock H. asinina populations derived from different areas was initially established from approximately 100 founders orig- investigated using the 3 microsatellite loci. inating in Cambodia (HACAMHE, N = 15) and Samet Is- land (HASAMHE, N = 10) and the second generation of H. asinina (HAPHIHE, N = 20) initially established from approximately 200 founders and maintained at the Aqua- MATERIALS AND METHODS culture Department, SEAFDEC, Philippines. Samples DNA Extraction Specimens of H. asinina were collected from 6 samples (Figure 1). Natural abalone include samples from Talibong Genomic DNA extracted from a single individual of H. Island (HATRAW, N = 28) located in the west of peninsular asinina (Gulf of Thailand origin) by a proteinase K phenol- Thailand, and Samet Island (HASAME, N = 12) and Cam- chloroform extraction method (Davis et al., 1986) was used 606 S. Tang et al. for construction of each partial genomic library. For Primer Design and Amplification of Microsatellites genotyping of abalone a Chelex-based extraction method (Walsh et al., 1991; Altschmied et al., 1997) was utilized Primer pairs to amplify microsatellite regions were de- (N = 108). signed using OLIGO 4.0 (National Biosciences; Table 1). Polymerase chain reaction (PCR) was carried out as Construction of H. asinina Partial Genomic described by Supungul et al. (2000). PCR products were Libraries analyzed on a 6% denaturing polyacrylamide gel at 50 W for 2.5 to 6 hours. After autoradiography allele sizes of Three partial genomic libraries were constructed: an AluI- each locus were determined by comparison with the M13 digested library (5 lgofH. asinina genomic DNA digested sequencing marker (Yanisch-Perron et al., 1985). Cross- with 25 U of AluIat37°C for 2 hours); a vortexed/sonicated species amplification for all loci was tested in H. ovina genomic library (1 lgofH. asinina genomic DNA vortexed (N = 5) and H. varia (N = 5) under different PCR for 15 minutes and subsequently sonicated in an Ultrasonic amplification conditions (annealing temperatures, BCGR 5139 bath for 1 hour); and a mixed-enzyme-digested MgCl2 concentrations, and thermal profiles) library (6 lgofH. asinina genomic DNA digested with 20 U each of AluI, HincII, and RsaI for 2 hours at 37°C). After Data Analysis agarose gel electrophoresis the resulting 300-bp to 800-bp DNA fragments were excised, eluted out from the gels, and The number of alleles per locus and observed, and expected further treated with T4 polynucleotide kinase and Klenow heterozygosity were calculated (Nei, 1987). The effective fragment according to conditions recommended by the number of alleles (Crow and Kimura, 1965) and allele fre- manufacturer (New England Biolabs). Approximately quencies at each locus in each sample were calculated. 150 ng of manipulated genomic DNA of H. asinina was Hardy-Weinberg equilibrium for each locus was examined ligated to 50 ng of dephosphorylated SmaI-digested pUC18 using the exact test (Rousset and Raymond, 1995). Linkage (Amersham Bioscience) overnight at 16°C. The ligation disequilibrium between loci in each sample and allele fre- mixture was electrotransformed into Escherichia coli XL1- quency distribution between possible sample combinations Blue (Dower et al., 1988). Recombinant clones were se- were compared using the Markov chain approach (Guo and lected on ampicillin agar plates according to standard Thompson, 1992). Significance of pairwise FST values (Weir protocol (Maniatis et al., 1982). and Cockerham, 1984) was evaluated. All the calculations described above were conducted using GENEPOP 2.0 Screening of Microsatellite-Containing Clones (Raymond and Rousset, 1995). The significance levels for multiple tests were adjusted following a sequential Bonfer- Transformed clones were transferred onto a piece of roni approach (Rice, 1989). Cavalli-Sforza and Edwards Whatman filter paper (#45). The filter paper was hybrid- chord genetic distance (Cavalli-Sforza and Edwards, 1967) 32 ized with the c- P labeled (GT)15 and (CT)15
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